Understanding the effects of HMW-GS and LMW-GS on processing quality using near-isogenic lines and development of functional markers for LMW-GS in wheat
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Understanding the effects of HMW-GS and LMW-GS on processing quality using near-isogenic lines and development of functional markers for LMW-GS in wheat
1. Understanding the effects of HMW-GS and LMW-
GS on processing quality using near-isogenic lines
and development of functional markers for LMW-
GS in wheat
Xianchun Xia, Zhonghu He
National Wheat Improvement Center,
Chinese Academy of Agricultural Sciences
xiaxianchun@caas.net.cn
3. Quality parameters
Flour yield, energy, extensograph maximum resistance,
farinograph stability time, and noodle viscoelasticity
Chinese steamed bread, fresh white Chinese noodle, pan
bread quality
glutenin macropolymers, dough properties
8. The effects of glutenin subunits on dough properties
Different letters indicate the significant difference at 5% probability level
9. The effects of glutenin subunits on pan bread quality
Different letters indicate the significant difference at 5% probability level
10. The effects of glutenin subunits on quantity of
gluten protein fractions
Different letters indicate the significant difference at 5% probability level
11. The effects of glutenin subunits on fresh white Chinese
noodle quality
Different letters indicate the significant difference at 5% probability level
12. The effects of glutenin subunits on northern style Chinese
steamed bread quality
Different letters indicate the significant difference at 5% probability level
13. The effects of combination of HMW-GS and LMW-GS on
dough properties
Trait Combination
ZSV Large 1/7+9/5+10/Glu-A3c/Glu-B3b/Glu-D3c
1/17+18/2+12/Glu-A3c/Glu-B3b/Glu-D3c
Small 1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c
1/7+9/2+12/Glu-A3c/Glu-B3c/Glu-D3c
ST Long 1/7+9/5+10/Glu-A3c/Glu-B3b/Glu-D3c
1/7+9/2+12/Glu-A3f/Glu-B3b/Glu-D3c
Short 1/7+9/2+12/Glu-A3c/Glu-B3c/Glu-D3c
1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c
Ext Good 1/7+9/2+12/Glu-A3c/Glu-B3i/Glu-D3c
1/7+9/2.2+12/Glu-A3c/Glu-B3b/Glu-D3c
Bad 1/7+9/2+12/Glu-A3c/Glu-B3c/Glu-D3c
1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c
14. The effects of combination of HMW-GS and LMW-GS on
quantity of gluten protein fractions
Trait Combination
Gli/Glu Low 1/7+9/2+12/Glu-A3c/Glu-B3g/Glu-D3c
1/7+9/3+12/Glu-A3c/Glu-B3b/Glu-D3c
High 1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c
1/7+9/2+12/Glu-A3c/Glu-B3c/Glu-D3c
%UPP High 1/7+9/5+10/Glu-A3c/Glu-B3b/Glu-D3c
1/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3d
Low 2*/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3c
1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c
15. The effects of combination of HMW-GS and LMW-GS on
processing quality
Trait Combination
PBTS High 1/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3f
1/7+9/2+12/Glu-A3c/Glu-B3g/Glu-D3c
Low 1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c
N/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3c
Noodle Good 1/7+9/2+12/Glu-A3c/Glu-B3d/Glu-D3c
1/7+9/2+12/Glu-A3c/Glu-B3f/Glu-D3c
viscoelasticity
Bad 1/7*+8/2+12/Glu-A3c/Glu-B3b/Glu-D3c
1/7+9/2+12/Glu-A3b/Glu-B3b/Glu-D3c
SBTS High 1/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3f
1/7+9/2+12/Glu-A3c/Glu-B3a/Glu-D3c
Low 1/7+9/2+12/Glu-A3c/Glu-B3g/Glu-D3c
1/7+9/2+12/Glu-A3c/Glu-B3d/Glu-D3c
17. Primers used for cloning Glu-B3 genes
Target Primer Sequence (5'→3') Reference Primer Ann temp
gene gene locationa (oC)
GluB3-1 LB1F GCACAAGCATCAAAACCAAGA AB262661 -13b 58
LB1R GACACTTTATTTGTCACCGCTG AB262661 1129
GluB3-2 LB2F AACCTAACGCATTGTACCAAAAATC AY542898 208 61
LB2R GGCGGGTCACACATGACA AY542898 1499
GluB3-3 LB3F CATCACAAGCACAAGCATCAA Y14104 472 58
LB3R CATATCCATCGACTAAACAAA AB119006 709
GluB3-4 LB4F CACCCTATACAAGGTTCCAAAAT Y14104 77 60
LB4R TATTTCCATAATTTAAACTAGTTTGT AB062852 +1346 b
18. Primers localization for cloning Glu-B3
a. LB1F-LB1R LB4F-LB4R LB2F-LB2R
b. LB3F-LB3R
Fig.1 Electrophoresis of the PCR products of the four gene-specific primer sets
on agarose gel. a GluB3-1, GluB3-4 and GluB3-2 genes, 1 Chinese Spring, 2
N1AT1B, 3 N1BT1A, 4 N1BT1D and 5 N1DT1B. b GluB3-3 gene, 1, 2, 5, 6, 7
1BL.1RS lines, 3 Aroona-B3c, 4 Aroona-B3d, 8 Aroona-B3h, 9 Aroona-B3i, 10
N1AT1B, 11 N1BT1A, 12 N1BT1D and 13 N1DT1B. M DNA Ladder 2000
(100, 250, 500, 750, 1,000 and 2,000 bp)
19. PCR amplification
Amplified the isogenic lines with
different Glu-B3 protein alleles.
Cloned 4 genes at Glu-B3 locus,
with 17 allelic variants.
20. Glu-B3 allelic variants
Allelic Accession No.
Gene variants Allele Size
GluB3-1 GluB3-11 a 1213 EU369699
GluB3-12 b 1228 EU369700
GluB3-13 e 1225 EU369701
GluB3-14 f 1222 EU369702
GluB3-15 g 1162 EU369703
GluB3-2 GluB3-21 a 1396 EU369704
GluB3-22 bef 1393 EU369721
GluB3-23 g 1393 EU369705
GluB3-3 GluB3-31 c 1290 EU369715
GluB3-32 d 1290 EU369716
GluB3-33 h 1290 EU369717
GluB3-34 i 1290 EU369718
GluB3-4 GluB3-41 acd 1636 EU369724
GluB3-42 b 1636 EU369719
GluB3-43 eh 1636 EU369727
GluB3-44 fg 1636 EU369729
GluB3-45 i 1615 EU369720
21. Relationship between Glu-B3 protein
alleles and gene haplotypes
Lines Allele GluB3 GluB3 GluB3- GluB3- GluB3- GluB3- GluB3- GluB3-
-11 -12 13 14 15 21 22 23
Aroona-B3a a + +
Aroona b + +
Aroona-B3c c
Aroona-B3d d
Cheyenne e + +
Aroona-B3f f + +
Aroona-B3g g + +
Aroona-B3h h
Aroona-B3i i
Lines Allele GluB3 GluB3 GluB3- GluB3- GluB3- GluB3- GluB3- GluB3- GluB3-
-31 -32 33 34 41 42 43 44 45
Aroona-B3a a +
Aroona b +
Aroona-B3c c + +
Aroona-B3d d + +
Cheyenne e +
Aroona-B3f f +
Aroona-B3g g +
Aroona-B3h h + +
Aroona-B3i i + +
23. Allele-specific PCR markers for the discrimination
of different Glu-B3 protein alleles
Marker Primer Sequence (5'→3') Target Fragment PCR condition
name set allele size
gluB3a SB1F GCACAAGCATCAAAACCAAGA a 1095 94℃/35s-55℃/35s-72℃/90s
SB1R TGGCACACTAGTGGTGGTC
gluB3b SB2F ATCAGGTGTAAAAGTGATAG b 1570 94℃/35s-56℃/35s-72℃/90s
SB2R TGCTACATCGACATATCCA
gluB3c SB3F CAAATGTTGCAGCAGAGA c 472 94℃/35s-56℃/35s-72℃/90s
SB3R CATATCCATCGACTAAACAAA
gluB3d SB4F CACCATGAAGACCTTCCTCA d 662 94℃/35s-58℃/35s-72℃/90s
SB4R GTTGTTGCAGTAGAACTGGA
gluB3e SB5F GACCTTCCTCATCTTCGCAa e 669 94℃/35s-58℃/50s-72℃/90s
SB5R GCAAGACTTTGTGGCATT
gluB3fg SB6F TATAGCTAGTGCAACCTACCAT fg 812 94℃/35s-62℃/35s-72℃/90s
SB6R CAACTACTCTGCCACAACG
gluB3g SB7F CCAAGAAATACTAGTTAACACTAGT g 853 94℃/35s-60℃/35s-72℃/90s
C
SB7R GTTGGGGTTGGGAAACA
gluB3h SB8F CCACCACAACAAACATTAA h 1022 94℃/35s-60℃/35s-72℃/90s
SB8R GTGGTGGTTCTATACAACGA
gluB3i SB9F As SB6F i 621 94℃/35s-58℃/35s-72℃/90s
SB9R TGGTTGTTGCGGTATAATTT
gluB3bef SB10F GCATCAACAACAAATAGTACTAGAA bef 750 94℃/35s-60℃/35s-72℃/90s
SB10R GGCGGGTCACACATGACA
24. STS markers
a b c d e f g h I
gluB3a
1095bp
gluB3b
1615bp
gluB3c
472bp
25. STS markers
a b c d e f g h I
gluB3d
662bp
gluB3e
669bp
gluB3bef
750bp
26. STS markers
a b c d e f g h I
gluB3fg
812bp
gluB3g
853bp
gluB3h
1022bp
gluB3i
621bp
27. Validation of molecular markers
Twenty wheat cultivars and 141 CIMMYT advanced
lines were tested with the markers. The results were
consistent with those detected by SDS-PAGE, except
for 4 lines.
Wang et al. 2009, Theor Appl Genet 118:525–539
28. Allele-specific PCR markers for the discrimination
of different Glu-A3 protein alleles
Marker Primer Sequence (5'→3') a Target Fragmen PCR condition
name set allele t
size
GluA3a LA1F AAACAGAATTATTAAAGCCGG a 529 94℃/35s-60℃/35s-72℃/90s
SA1R GGTTGTTGTTGTTGCAGCA
GluA3b LA3F TTCAGATGCAGCCAAACAA b 894 94℃/35s-60℃/35s-72℃/90s
SA2R GCTGTGCTTGGATGATACTCTA
GluA3ac LA1F AAACAGAATTATTAAAGCCGG acb 573 94℃/35s-60℃/35s-72℃/90s
SA3R GTGGCTGTTGTGAAAACGA
GluA3d LA3F TTCAGATGCAGCCAAACAA d 967 94℃/35s-60℃/35s-72℃/90s
SA4R TGGGGTTGGGAGACACATA
GluA3e LA1F AAACAGAATTATTAAAGCCGG e 158 94℃/35s-60℃/35s-72℃/90s
SA5R GGCACAGACGAGGAAGGTT
GluA3f LA1F AAACAGAATTATTAAAGCCGG f 552 94℃/35s-60℃/35s-72℃/90s
SA6R GCTGCTGCTGCTGTGTAAA
GluA3g LA1F AAACAGAATTATTAAAGCCGG g 1345 94℃/35s-60℃/35s-72℃/90s
SA7R AAACAACGGTGATCCAACTAA
29. Glu-A3 marker
A3a A3b A3c A3d A3e A3f A3g
AF33-A3A3R
GluA3a
AF63-A6B3R
GluA3b
AF14-A3AC2R
GluA3ac
Wang et al. 2010, J. Cereal Sci 51:305-312
30. Glu-A3 marker
A3a A3b A3c A3d A3e A3f A3g
AF63-A6D2R
GluA3d
Af522-A5d1r
GluA3d
AF14-A3E3R
GluA3e
Wang et al. 2010, J. Cereal Sci 51:305-312
31. Glu-A3 marker
A3a A3b A3c A3d A3e A3f A3g
A3F1F-A3F3R
GluA3f
AF33-A1G1R
GluA3g
Wang et al. 2010, J. Cereal Sci 51:305-312
33. Glu-A3 multiplex PCR
a
GluA3b GluA3d
GluA3be + GluA3df
967bp
894bp
GluA3f
552bp
GluA3e
158bp
b
GluA3bf + GluA3dg GluA3g
GluA3b GluA3d 1345bp
967bp
GluA3f
894bp
552bp
Wang et al. 2010, J Cereal Science 51:305-312
34. Acknowledgement
Ken Shepherd, SARDI Grain Quality Research Laboratory
Marie Appelbee, SARDI Grain Quality Research Laboratory
Rudi Appels, University of Murdoch
Wujun Ma, University of Murdoch
Roberto Peñ CIMMYT
a,
Aimin Zhang, Institute of Genetics, CAS
Peiyuan Mu and Zheru Fan for their assistance in the field trials
Xianlin Zhao
Linhai Wang
Xiaofei Zhang
Hui Jin
35. These studies were supported by
the National Basic Research Program (2009CB118300),
National Natural Science Foundation of China (30830072),
International Collaboration Project from Ministry of
Agriculture (2011-G3)